Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
  • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
  • A01H1/08—Methods for producing changes in chromosome number
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
  • C12N15/823—Reproductive tissue-specific promoters
  • C12N15/8231—Male-specific, e.g. anther, tapetum, pollen
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
  • C12Y301/01002—Arylesterase (3.1.1.2)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/04—Phosphoric diester hydrolases (3.1.4)
  • C12Y301/04004—Phospholipase D (3.1.4.4)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• the present invention relates to the field of simplification of laborious breeding programs using molecular biological methods, marker technology and genetic engineering.
• sorghum sorghum plants are provided which are capable of inducing haploidy through modifications in the genome concerning a preferably pollen-specific expressed patatinous phospholipase, thereby producing haploid offspring and can be produced in a short time by chromosome duplication inbred lines for hybrid breeding.
• sorghum sorghum plants which have mutations in the patatin phospholipase, methods for producing and identifying these mutations or the mutated plant and the corresponding nucleic acid molecule which encodes the mutated patatin phospholipase and vectors and host cells in particular plant cells containing the nucleic acid molecule and plants generated from such plant cells which are capable of producing haploidy and their progeny, crossbred products, inbred lines and their respective plant parts and products.
• the present invention provides methods for the production and identification of transgenic and non-transgenic plant haploid inducers as well as the corresponding plants which have obtained the property of haploid induction or whose induction performance has been improved. Furthermore, the invention also includes seeds or offspring, organs, plant parts, tissues or cells of the plant according to the invention and their use.
• the present invention therefore relates to the embodiments listed in the following items [1] to [29] and illustrated in the examples.
• Sorghum millet which is capable of inducing haploidy, characterized in that the plant has one or more modifications relating to an endogenous patatinous phospholipase, which is preferably expressed pollen-specific.
• [7] Plant according to any one of [4] to [6], characterized in that the one or more mutations to an amino acid exchange at the amino acid position 59, 162 and / or 291 according to SEQ ID NO: 3, and / or to a Stop codon at amino acid position 372 according to SEQ ID NO: 3 lead.
• nucleotide sequence encoding the coding sequence of the DNA sequence according to SEQ ID NO: 1 (derivable from the corresponding cDNA according to SEQ ID NO: 2) or a DNA sequence which is at least 80% identical is to SEQ ID NO: 1, in which there is at least one nucleotide exchange resulting in an amino acid substitution, wherein one or more nucleotides at nucleotide positions 421-423, 815-817, 1420-1422 and / or 1663 -1665 according to SEQ ID NO: 1 (corresponding to nucleotide positions 175-177, 484-486, 871-873 and / or 1114-1116 of SEQ ID NO: 2); (iii) an amino acid sequence according to SEQ ID NO: 6, 9 or 12 comprises; or
• Nucleic acid molecule according to [10] characterized in that its presence in a plant, preferably in the absence of a wild type patatin phospholipase, results in the plant being able to induce haploidy.
• PCR polymerase chain reaction
• Vector preferably a plant vector comprising a nucleic acid molecule according to one of [10] to [12], or an expression cassette defined in [4].
• Host cell preferably a plant cell containing a nucleic acid molecule according to one of [10] to [12], an expression cassette defined in [4], a vector according to [13] or the nucleic acid molecule according to any of [10] to [12] as transgene , optional under the Control of a heterologous promoter, preferably a pollen-specific promoter.
• a method for obtaining a plant capable of inducing haploidy or having an increased rate of induction against the wild type comprising the following steps:
• Has mutations which correspond to one or more of the mutations identified in [4] to [8] and / or which lead to aspartic acid (D) at position 75, to glycine (G) at position 79, and / or to the position 203 proline (P) of the amino acid sequence according to SEQ ID NO. 3 is replaced by another amino acid, preferably by asparagine (N) at position 75, arginine (R) at position 79 and / or leucine (L) at position 203 or which correspond to these, in plant cells, or introduction of the expression cassette defined in [4] into plant cells; and (ii) regenerating a plant, for example a transgenic plant, from the plant cells (i).
• the endogenous DNA sequence or the nucleic acid molecule is a coding nucleotide sequence which corresponds to the in Sorghum sorghum (SEQ ID NO: 3), SEQ ID NO: 18 for sunflower (Helianthus annuus), SEQ ID NO: 21 for barley (Hordeum vulgare) or SEQ ID NO: 24 or 27 for beta vulgaris (eg sugar beet) shown amino acid sequences.
• Method according to [16], wherein the introduction of the nucleic acid molecule can be carried out, for example, by Agrobacterium transformation, homologous recombination, for example by means of CRISPR / Cas or CRISPR / Cpfl and repair template, and the mutagenizing chemical and physical mutagenesis, TILLING, directed mutagenesis, for example by use of zinc finger nucleases, Transcription Activator-like Effector (TALE) nucleases, meganucleases, and the CRISPR / Cas and CRISPR / Cpfl systems, respectively.
• Agrobacterium transformation homologous recombination
• homologous recombination for example by means of CRISPR / Cas or CRISPR / Cpfl and repair template
• TILLING mutagenizing chemical and physical mutagenesis
• directed mutagenesis for example by use of zinc finger nucleases, Transcription Activator-like Effector (TALE) nucleases, meganuclea
• a method for obtaining a haploid plant comprising the following steps:
• a method for obtaining diploid plants comprising the following steps:
• a method of producing hybrid plants comprising the following steps:
• nucleic acid according to [12] as a molecular marker for detecting a mutation in the patatin phospholipase gene.
• To confer the property of a haploid inducer” or “to confer the property of a haploid inducer” or “to be able to induce haploidy” means a similar expression to that of a plant by use of a nucleic acid of the invention or by modification of the genome, in particular by mutation a patatinous phospholipase capable of producing fertilized seeds or embryos having a simple (haploid) set of chromosomes from a cross with a plant of the same genus, preferably the same species, which does not possess the property of a haploid inducer one Haploid inductor, expressed as the absolute haploid induction rate, means that at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0, 8%, 0.9% or 1%, preferably at least 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%, particularly preferably at least 6% , 7%, 8%, 9%, 10%, 11%, 12%, 1
• a "functional fragment" of a nucleotide sequence means a portion of a nucleotide sequence which has the identical or comparable functionality as the total nucleotide sequence from which the functional fragment is derived.
• the functional fragment may have a nucleotide sequence which is over a length of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94% 96%. , 97%, 98% or 99% is identical or homologous with the total nucleotide sequence.
• a "functional fragment" of a nucleotide sequence may also mean a portion of a nucleotide sequence which alters the functionality of the entire nucleotide sequence, for example in the course of posttranscriptional or transcriptional gene silencing.
• the functional fragment of a nucleotide sequence may be at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, preferably at least 30, 35, 40, 45, 50, 60, 70, 80 , 90, 100, 120 or 140, more preferably at least 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000 consecutive nucleotides of the total nucleotide sequence.
• a “functional part" of a protein means a portion of a protein or a portion of the amino acid sequence that encodes the protein, which portion can perform the identical or comparable functionality as the total protein in a plant cell.
• a functional part of a protein has a length of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94% 96%, 97%, 98 % or 99% of an amino acid sequence identical or conservative considering conservative and semi-conservative amino acid substitutions as the protein from which the functional part is derived.
• Haploid Inductor also means an in vivo haploid inductor.
• heterologous means that the introduced polynucleotide, for example, originates from one cell or organism with another genetic background of the same species or species, or is homologous to the prokaryotic or eukaryotic host cell, but in a different genetic environment is localized and thus different from any naturally occurring corresponding polynucleotide.
• a heterologous polynucleotide may be present in addition to a corresponding endogenous gene.
• Hybridization or “hybridization” is understood to mean a process in which a single-stranded nucleic acid molecule attaches to a largely complementary nucleic acid strand, ie, bases pairings with it. Standard methods for hybridization are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001. Preferably, it is understood that at least 80% or 85%, preferably at least 90%, 91%, 92%, 93%, 94% or 95%, particularly preferably at least 96%, 97%, 98% or 99% of the bases of Nucleic acid molecule enter into a base pairing with the largely complementary nucleic acid strand.
• stringency refers to the hybridization conditions. High stringency is given when base pairing is difficult, low stringency when base pairing is facilitated.
• the stringency of the hybridization conditions depends, for example, on the salt concentration or ionic strength and the temperature. In general, the stringency can be increased by increasing the temperature and / or lowering the salt content.
• stringent hybridization conditions are meant those conditions in which a hybridization occurs predominantly only between homologous nucleic acid molecules and homologs.
• hybridization conditions does not only refer to the conditions prevailing in the actual attachment of the nucleic acids, but also to the conditions prevailing during the subsequent washing steps.
• Stringent hybridization conditions are, for example, conditions under which predominantly only those nucleic acid molecules which have at least 80%, at least 85%, at least 90% or at least 95% sequence identity hybridize.
• Stringent hybridization conditions are, for example: hybridization in 4 x SSC at 65 ° C followed by multiple washes in 0.1 x SSC at 65 ° C for a total of about 1 hour.
• the term "stringent hybridization conditions" as used herein may also mean hybridization at 68 ° C in 0.25 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA and 1% BSA for 16 hours and then washing twice with 2x SSC and 0.1% SDS at 68 ° C.
• hybridization takes place under stringent conditions.
• Increase the induction performance of a haploid inducer or “increase the induction performance of a haploid inducer” or similar terms mean that the haploid induction rate of a plant exhibiting the property of a haploid inducer is increased.
• the number of fertilized seeds having a haploid set of chromosomes resulting from crossing the haploid inducer with a plant of the same genus, preferably the same species not having the property of a haploid inducer may be at least 0.1%, 0.2 %, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%, preferably at least 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%, and most preferably at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30 % or 50% higher than the number of haploid fertilized seeds obtained without the use of the nucleic acid or without modification of the genome, in particular without mutation of a patatin phospholipase according to the present invention, ie the haploid induction rate can be at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%, preferably at least 1.5%,
• “Complementary" nucleotide sequence with respect to a nucleic acid in the form of a double-stranded DNA means that the second DNA strand complementary to the first DNA strand has the nucleotides corresponding to the bases of the first strand according to the base-stranding rules according to the Watson-Crick rules.
• a “molecular marker” is a nucleic acid that is polymorphic in a plant population and used as a reference or landmark.
• a marker for detecting a recombination event should be capable of monitoring differences or polymorphisms within a plant population. Thus, such a marker is capable of detecting and distinguishing various allelic states (alleles).
• the term "molecular marker” also refers to nucleotide sequences which are complementary or at least largely complementary or homologous to genomic sequences, for example nucleic acids, which are used as probes or primers.
• markers in the specification may also mean a specific chromosome position in the genome of a species where a specific marker (eg, SNP) can be found.
• a "plant” within the meaning of the invention may, unless stated otherwise, be of any species of the dicotyledonous and monocotyledonous plants. Preference is given to plants in agriculture or horticulture or for the production of bioenergy (bioethanol, biogas, etc.).
• Plant “organs” mean, for example, leaves, stem axis, stem, roots, vegetative buds, meristems, embryos, anthers, ovules, seeds or fruits, especially seeds.
• plant part or “plant parts” includes, but is not limited to, the shoot axis or stem, leaves, flowers, inflorescences, roots, fruits and seeds as well as the pollen.
• Herbal "parts” also mean an association of several organs, e.g. a flower or seed, or part of an organ, e.g. a cross section of the stem axis.
• Herbal "tissues” are, for example, callus tissue, storage tissue, meristematic tissues, leaf tissue, shoot tissue, root tissue, plant tumor tissue or reproductive tissue and the formation tissue, ground tissue (the so-called parenchyma), Leitgewebe, Festistrsgewebe and the cover tissue (the so-called epidermis).
• the fabric is not limited by this listing.
• plant “cells” are meant, for example, isolated cells with a cell wall or aggregates thereof or protoplasts.
• a “promoter” is an untranslated DNA segment, typically upstream of a coding region, which includes the binding site for the RNA polymerase and initiates transcription of the DNA.
• a promoter also contains other elements that act as regulatory gene expression genes (e.g., cis-regulatory elements).
• a “core or minimal promoter” is a promoter that has at least the primitives needed for transcription initiation (e.g., TATA box and / or initiator).
• modifications refers to a nucleotide sequence which influences the specificity and / or the expression level, for example by the regulatory sequence mediating a specific tissue specificity.
• a regulatory sequence may be upstream of the transcription initiation point of a Minimal promotors, but also downstream thereof, such as in a transcribed but untranslated leader sequence or located within an intron.
• a “transgenic plant” refers to a plant in whose genome at least one polynucleotide, preferably a heterologous polynucleotide, is integrated.
• the polynucleotide is stably integrated, which means that the integrated polynucleotide is stably maintained in the plant, expressed and can be stably inherited to the offspring.
• the stable introduction of a polynucleotide into the genome of a plant also includes integration into the genome of a plant of the previous parental generation, wherein the polynucleotide can be stably inherited.
• heterologous means that the introduced polynucleotide, for example, originates from one cell or organism with another genetic background of the same species or species, or is homologous to the prokaryotic or eukaryotic host cell, but is then located in a different genetic environment and thus differs from any naturally occurring corresponding polynucleotide.
• a heterologous polynucleotide may be present in addition to a corresponding endogenous gene.
• Suitable for use as a haploid inducer or “capable of inducing haploidy” means that a plant is capable of fertilized seeds having a simple (haploid) set of chromosomes from a cross with a plant of the same genus, preferably the same Species, which does not have the property of a Haploideninduktors bring forth.
• haploid inducer expressed as the absolute haploid induction rate, means that at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7% , 0.8%, 0.9% or 1%, preferably at least 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%, more preferably at least 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%, or even more preferably at least 20%, 25%, 30%, 35%, 40% , 45% or 50% of the fertilized seeds have a haploid set of chromosomes.
• Figure 1 Sequence alignment (CLUSTAL O 1.2.4) of the amino acid sequences of the wild-type
• Sorghum sorghum patatin phospholipase (Sorghum_PPL_AA, SEQ ID NO: 3), the sorghum sorghum patatin phospholipase with an exchange of the Amino acid arginine (R) by glutamine (Q) at amino acid position 59 (Sorghum_PPL_R59Q, SEQ ID NO: 6), sorghum sorghum patatin phospholipase with an exchange of the amino acid valine (V) with isoleucine (I) at amino acid position 162 ( Sorghum_PPL_V162I, SEQ ID NO: 9), the sorghum sorghum patatin phospholipase with replacement of the amino acid serine (S) by leucine (L) at amino acid position 291 (Sorghum_PPL_S291L, SEQ ID NO: 12) and patatin phospholipase Sorghum millet with a replacement of the amino acid glutamine (Q) with
• Figure 2 Sequence alignment (CLUSTAL O 1.2.4) of the amino acid sequences of the wild-type
• Sorghum sorghum patatin phospholipase (Sorghum bicolor_PPL_AA, SEQ ID NO: 3), barley patatin phospholipase (Hordeum vulgare_PPL_AA, SEQ ID NO: 21), sunflower patatin phospholipase (Helianthus annuus_PPL_AA, SEQ ID NO: 1). 18), patatin phospholipase 1 from sugar beet (Beta vulgaris_PPLI_AA, SEQ ID NO: 24) and patatin phospholipase 2 from sugar beet (Beta vulgaris phospholipase 2_AA, SEQ ID NO: 27). The amino acid positions which are the functional domain of phospholipase are shown in bold.
• the present invention provides a plant of the genus Sorghum, hereinafter also referred to as sorghum sorghum, which is capable of inducing haploidy.
• sorghum sorghum which is capable of inducing haploidy.
• the plant is capable of producing fertilized seeds or embryos having a simple (haploid) set of chromosomes from a cross with a plant of the same genus, preferably the same species, which does not possess the property of a haploid inducer.
• the inventors have been able to provide an efficient system for breeding sorghum sorghum plants, because by inoculation lines, ie homozygous parent and father lines for hybrid breeding can be generated by chromosome doubling in the haploid offspring.
• the sorghum sorghum plants according to the invention are characterized in that they have one or more modifications which relate to the patatinous phospholipase, which either mediates the haploid induction property or also improves a naturally present ability for haploid induction or increases the induction performance.
• the modification concerning the patatin phospholipase is the cause of the suitability of the sorghum sorghum plant according to the invention as a haploid inducer in breeding crops.
• the patatin phospholipase in sorghum sorghum is pollen-specifically expressed and therefore presumably has an influence on the pollen tube growth or the interaction between the gametophytes.
• the sorghum sorghum plant according to the invention is preferably characterized in that the patatin phospholipase is pollen-specifically expressed and / or has an influence on the pollen tube growth or the interaction between the gametophytes.
• the patatin phospholipase is encoded by the nucleotide sequence according to SEQ ID NO: 1 or 2 or by a nucleotide sequence which is at least 80%, preferably 85%, more preferably 90%, even more preferably 95% and most preferably 99% identical to SEQ ID NO: 1 or 2, is encoded or is encoded by a nucleotide sequence which hybridizes with the complementary sequence to the nucleotide sequence according to SEQ ID NO: 1 or 2 under stringent conditions, or in SEQ ID No. 3 amino acid sequence or a homologous amino acid sequence.
• the sorghum sorghum plant according to the present invention comprises plants of any species of the genus Sorghum, in particular of the species Sorghum bicolor, Sorghum sudanense and Sorghum bicolor x Sorghum sudanense or their hybrids and all the varieties derived therefrom. Accordingly, it is reasonable to assume that in the course of speciation and variety breeding, the nucleotide sequence and, correspondingly, the amino acid sequence of the patatin phospholipase have changed.
• homologous means that the genes in question (from two different plant species or varieties) have essentially the same function and a common precursor, and therefore typically exhibits a significant identity in its nucleic acid or encoded amino acid sequence, which is preferably at least 80%.
• a “homologue” is understood to mean a protein of the same phylogenetic origin, understood by an “analog” to be a protein which has the same function but has a different phylogenetic origin, and an "orthologue” to a protein from another species understood that performs the same function and understood by a "paralogue” a protein that has been created by duplication within a species, this copy either retains the same protein function, their expression pattern changes, but does not change the function, their protein function or the separates original gene function between both copies.
• an amino acid sequence in the sorghum sorghum patatin phospholipase (SEQ ID NO: 3) in sorghum sorghum leads to an amino acid sequence Haploid induction rate of sometimes more than 1.5%. It can be assumed that a further increase in the induction rate can result from further amino acid substitutions, which leads to a further modification of the coding sequence of the patatin phospholipase.
• sorghum sorghum plants comprising one or more modifications or mutations relating to patatin phospholipase are included in the present invention.
• the abovementioned modifications which relate to patatinous phospholipase are preferably characterized in that they are mutations which result in the endogenous DNA sequence coding for the patatinous phospholipase, in one or more amino acid substitutions or in the production a stop codon.
• a mutation means a modification at the DNA level, ie a change in genetics and / or epigenetics.
• a change in genetics may be the replacement of at least one nucleobase in the endogenous DNA sequence or in a regulatory sequence of the endogenous DNA sequence. If such nucleobase exchange takes place e.g.
• a change in genetics is the deletion of nucleotides in the regulatory sequence and / or the endogenous DNA sequence and the addition of nucleotides in the regulatory sequence and / or the endogenous DNA sequence.
• a change in the epigenetics can be done for example by an altered methylation pattern of the DNA.
• stop codons in the functional domains of a protein usually leads to a loss of the function of the protein, whereas the generation of a stop codon after the functional domain could also lead to an increase in the activity or stabilization of the protein.
• mutations which are preferably amino acid substitutions, there may be an increase in the activity of the protein, for example, by optimization of the sequence or equally well to inhibition or loss of activity.
• mutations in the promoter region can lead to a change in the expression of the gene.
• the mutation described allows the biological activity of the patatinous phospholipase to be altered so that its original function in pollen is no longer exercised to the same extent as is the case in the wild-type sorghum sorghum.
• the overexpression of the gene could also inhibit, prevent or reduce the correct formation, folding and / or stability of the patatin phospholipase.
• a functional patatin phospholipase could occur, for example, through mutations in the promoter region, to the formation of a less active, inactive, or unstable form of the patatinous phospholipase, e.g. Generation of a stop codon or by amino acid substitutions in the functional domain, which in turn could lead to faulty fertilization.
• the localization of the patatin phospholipase could be altered by the described mutations, so that it is no longer pollen-specifically expressed, for example.
• sorghum millet plants comprising one or more insertions of an expression cassette comprising a nucleic acid molecule encoding the patatin phospholipase represented by the nucleotide sequence of SEQ ID NO: 1 or 2 or by a nucleotide sequence corresponding to at least 80% are also included in the present invention %, preferably 85%, more preferably 90%, even more preferably 95%, and most preferably 99% is identical to SEQ ID NO: 1 or 2, or encoding a functional part of this patatin phospholipase, or having a nucleic acid molecule a nucleotide sequence which hybridizes with the complementary sequence to the nucleotide sequence according to SEQ ID NO: 1 or 2 under stringent conditions, or which comprises a nucleic acid molecule encoding the patatin phospholipase having an amino acid sequence according to SEQ ID NO: 3 or a homologous amino acid sequence or comprises a functional part thereof, and operatively linked to
• sorghum sorghum plants in which the expression of the patatin phospholipase is partially or completely inhibited or a reduced amount of the patatin phospholipase protein is present or no functional patatin phospholipase is formed.
• the present invention also includes sorghum sorghum plants in which the expression of said patatinous phospholipase is partially or completely inhibited by an RNAi approach (Fire et al., Nature 391 (1998), 806-811).
• the plant according to the invention is further characterized in that it comprises one or more insertions of an expression cassette which comprises a promoter and optionally a terminator which is operatively linked to a nucleic acid molecule which codes for a dsRNA comprising at least 19 or 20, preferably at least 21, 22, 23, 24 or 25, particularly preferably at least 30, 35, 40, 45 or 50, and particularly preferably at least 100, 200, 300 or 500 nucleotides which are complementary to a partial sequence of the nucleotide sequence according to SEQ ID NO.
• an expression cassette which comprises a promoter and optionally a terminator which is operatively linked to a nucleic acid molecule which codes for a dsRNA comprising at least 19 or 20, preferably at least 21, 22, 23, 24 or 25, particularly preferably at least 30, 35, 40, 45 or 50, and particularly preferably at least 100, 200, 300 or 500 nucleotides which are complementary to a partial sequence of the nucleotide sequence according to SEQ ID NO.
• nucleotide sequence which has at least 80%, preferably 85%, more preferably 90%, even more preferably 95% and most preferably 99% identical to a partial sequence of the nucleotide sequence according to SEQ ID NO: 1 or 2 , includes.
• the present invention also sorghum sorghum plants in which there is a knock-out of patatinous phospholipase by mutation.
• Suitable promoters useful in the expression cassettes may be promoters which are constitutively induced (e.g., 35S promoter from the cauliflower mosaic virus (Odell et al., Nature 313: 810-812, 1985)) those promoters which are development-specific (eg, flower-specific promoters) or tissue-specific, in particular those which are specifically active in pollen (examples: Chen et al., Molecular Biology Reports 37 (2010), 737-744, Zhao et al., Planta 224 (2006), 405-412 or Twell et al., Genes & Development 5 (1991), 496-507)
• Suitable promoters may also be synthetic or chimeric promoters, which do not occur in nature, are composed of several elements and a minimal promoter and upstream of the minimal promoter have at least one ds-regulatory element which serves as a binding site for specific transcription factors chimeric promoters can the desired Spezi and are induced or repressed by different factors.
• promoters can be found in Gurr and Rushton (TRENDS in Biotechnology 23 (2005), 275-282) or Venter (Trends in Plant Science 12 (2007), 118-1249
• a suitable terminator is, for example, the nos terminator (Depicker et al., Journal of Molecular and Applied Genetics 126 (1982), 561-573). Promoters and other transcriptional regulatory elements are well known and are available to those skilled in the art, see, for example, WO 00/75359 at page 23, line 5 to page 24, line 17.
• the present invention provides sorghum sorghum plants having one or more mutations resulting in an amino acid substitution in the region of amino acid positions 37 to 240 of SEQ ID NO: 3, which preferably corresponds to the functional domain of patatin phospholipase.
• the one or more mutations in the range of amino acid positions 40 to 93 or 135 to 204 preferably in the range of amino acid positions 53-85 or 150-192, more preferably in the range of amino acid positions 55-75 or 157- 167 to an amino acid exchange.
• sorghum sorghum plants results in a haploid induction rate of more than 1.5%.
• a sorghum millet haploid inducer could be generated by a mutation in the nucleotide sequence of SEQ ID NO: 1 that resulted in a stop codon at amino acid position 372 of SEQ ID NO: 3. This mutation replaced the amino acid glutamine (Q) at position 372 with a stop codon.
• mutants generated by transposons can be detected by using transposon specific primers and target gene specific primers in PCR over the entire population and subsequent sequencing of PCR products.
• primers are also covered by the present invention.
• the present invention also relates to molecular markers which detect the presence or absence of a mutation in the endogenous DNA sequence or in a regulatory sequence of the endogenous DNA sequence. For example, such markers are based on an SNP and are specific for the mutation (examples: KASPar or TaqMan markers).
• the identification of a plant in step (b) may also be carried out by testing the induction performance as described in pending Example 1.
• the present invention also relates to a method for identifying a plant according to the invention by detecting the mutation in the patatin phospholipase gene or by detecting a marker allele which is coupled to the mutation, preferably using molecular markers described above.
• An example of a plant produced and identified by such a method is the sorghum millet of the invention.
• the present invention further relates to a plant producible or produced by the above method, or a part thereof, wherein a part of a plant comprises a fertilized or unfertilized seed, an embryo, a pollen, a tissue Organ or a plant cell, wherein the fertilized or unfertilized seed, the embryo or the pollen are produced on the transgenic plant and in whose genome the at least one mutation is present.
• the present invention also includes a descendant of the plant which has the at least one mutation and is suitable for use as a haploid inducer.
• the method can be applied to any plant containing a patatinous phospholipase and thus given the property of haploid induction.
• this plant is Sorghum sorghum, sunflower, barley, sugar beet, rye, wheat or potato.
• the present invention also relates to a method of producing a transgenic plant suitable for use as a haploid inducer.
• the method may include the following steps:
• nucleic acid molecule according to the invention which encodes the patatin phospholipases described above and has therein one or more mutations leading to one or more of the described amino acid substitutions or to the generation of a stop codon, preferably to one or more amino acid substitutions selected from S291L , R59Q, V162I and Q372 stop according to the amino acid sequence SEQ ID NO. 3, introducing a nucleic acid molecule having one or more mutations resulting in one or more amino acid substitutions in sequences of plant patatin phospholipases comprising the amino acid substitutions S291L, R59Q, VI 621 and Q372 stop according to the amino acid sequence SEQ ID NO.
• the present invention also relates to a transgenic plant which is produced or produced by this method, or a part of this plant, wherein a part of a plant is a fertilized or unfertilized seed, an embryo, a pollen, a tissue, an organ or a can be plant cell, wherein the fertilized or unfertilized seed, the embryo or the pollen are produced on the transgenic plant and in whose genome the introduced nucleic acid is integrated as a transgene or the vector.
• the present invention also includes a descendant of the transgenic plant having the introduced nucleic acid as a transgene and for use as a haploid inducer suitable is.
• the plant suitable for use as a haploid inducer is used as a pollen parent and crossed with a seed stainer of the same genus, preferably of the same species.
• the plant suitable for use as a haploid inducer can be used as a seed and crossed with a pollen parent of the same genus, preferably the same species.
• Both crossing partners in step (a), ie Saateiter and Pollenelter, can also be the same individual. Then the crossing step represents a selfing.
• the haploid plants from (a) are treated with the cell division inhibitor colchicine. This leads to a doubling of the chromosomes.
• the skilled person is aware of this process and it is described, for example, in Segui-Simarro and Nuez, Cytogenetic and Genome Research 120 (2008), 358-369.
• the double-haploid plants are pure and by crossing two pure-leaf plants, the known heterosis effect occurs, which leads to a particularly pronounced performance of hybrid plants. Accordingly, the hybrid plants obtained by such a method are also the subject of the present invention.
• a gene could be found, in particular the gene of the patatin phospholipase with one or more mutations which were able to confer on the plant the property of a haploidy inducer and an induction capacity of at least 0.4% up to 1 , 5% or more, so that an efficient and therefore economically applicable system for the production of haploid and double haploid sorghum millet plants for hybrid breeding could be provided.
• the inventive method for producing such Haploideninduktoren and the identification of patatinous phospholipases in other crops, this system can also be transferred to this.
• a sorghum millet was found which was able to induce haploidy with an efficiency of about 1%.
• search Based on the genetic basis that mediates this trait in the identified sorghum herb, it has focused on various genes as potential targets. The selection of genes has been made based on the fact that they are preferentially expressed in reproductive organs of the plant and play in some way a role in the fertilization, because by incorrect or incomplete fertilization, for example by a lack or incorrect transport of the generative cells to the female Ovulation or by acting on the energy metabolism of the pollen, it can lead to chromosome elimination, resulting in a haploid chromosome set.
• a mutated gene was then identified which was identified as patatin phospholipase in sorghum by means of the bioinformatic methods BLASTP and Synteny study (Altschul et al, Nucleic Acid Res. 25 (1997), 3389-3402) and whose nucleotide and amino acid sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 3, respectively, and has been identified as a pollen-specific expressed patatin phospholipase by RNA sequencing (RNASeq).
• the mutant gene had a point mutation in the nucleotide sequence of the patatin phospholipase (see SEQ ID NO: 10), which caused an amino acid change from serine to leucine at position 291 (see SEQ ID NO: 12).
• the point mutation in the nucleotide sequence of the patatin phospholipase was identified using the PCR method with the primers according to SEQ ID NOs: 44 and 45.
• the induction property of the sorghum sorghum plants described above can be attributed to the described mutations in the patatin phospholipase, which usually leads to a change in the biological activity of these.
• the biological activity can be changed not only by introducing the described mutations but also by numerous other genetic engineering methods.
• amino acid substitutions at position 75 are replaced by asparagine (N) (D75N)
• at position 79 glycine (G) is replaced by arginine (R) (G79R)) and / or at position 203 (proline (P) is replaced by leucine (L) (P203L)) of the amino acid sequence according to SEQ ID No. 3 can induce induction of haploids both in sorghum and in other cultivars

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